The human cut homeodomain protein represses transcription from the c-myc promoter.

Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene.

DNA-binding specificity of the cut repeats from the human cut-like protein.

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy.

By analogy with other homeodomain proteins conserved in evolution, mammalian Cut proteins are believed, as in Drosophila melanogaster, to play an important role in determining cell type specificity in several tissues. At the molecular level, Cut proteins appear to serve as transcriptional repressors. In this study, we have examined the mechanism by which the human Cut (hCut) protein down-regulates gene expression. The homeodomain and the three regions called Cut repeats are evolutionarily conserved and were previously shown to function as DNA binding domains. The carboxy-terminal region, although it does not show amino acid sequence homology per se, in all cases is enriched in alanine and proline residues, a distinctive feature of some transcriptional repression domains. Our results reveal two distinct modes of repression: competition for binding site occupancy and active repression. On one hand, the composite DNA binding domain formed by Cut repeat 3 and the Cut homeodomain was shown to bind to CCAAT and Sp1 sites within the tk gene promoter and to reduce gene expression, presumably by preventing activation by the corresponding transcription factors. On the other hand, the carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner. We have further narrowed this activity to two subdomains that can independently repress activated transcription. Finally, we present a model to illustrate the two mechanisms by which Cut proteins repress gene expression.

S phase-specific proteolytic cleavage is required to activate stable DNA binding by the CDP/Cut homeodomain protein.

The CCAAT displacement protein (CDP), the homologue of the Drosophila melanogaster Cut protein, contains four DNA binding domains that function in pairs. Cooperation between Cut repeat 3 and the Cut homeodomain allows stable DNA binding to the ATCGAT motif, an activity previously shown to be upregulated in S phase. Here we showed that the full-length CDP/Cut protein is incapable of stable DNA binding and that the ATCGAT binding activity present in cells involves a 110-kDa carboxy-terminal peptide of CDP/Cut. A vector expressing CDP/Cut with Myc and hemagglutinin epitope tags at either end generated N- and C-terminal products of 90 and 110 kDa, suggesting that proteolytic cleavage was involved. In vivo pulse/chase labeling experiments confirmed that the 110-kDa protein was derived from the full-length CDP/Cut protein. Proteolytic processing was weak or not detectable in G(0) and G(1) but increased in populations of cells enriched in S phase, and the appearance of the 110-kDa protein coincided with the increase in ATCGAT DNA binding. Interestingly, the amino-truncated and the full-length CDP/Cut isoforms exhibited different transcriptional properties in a reporter assay. We conclude that proteolytic processing of CDP/Cut at the G(1)/S transition generates a CDP/Cut isoform with distinct DNA binding and transcriptional activities. These findings, together with the cleavage of the Scc1 protein at mitosis, suggest that site-specific proteolysis may play an important role in the regulation of cell cycle progression.

A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

Transcriptional activation of the mouse mdr3 gene coincides with the appearance of novel transcription initiation sites in multidrug-resistant P388 tumor cells.

In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene. In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification. The mechanism underlying mdr3 overexpression in these cells was investigated. Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene. Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1). Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon. In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2. Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Plasminogen activator inhibitor 1 messenger RNA expression and molecular evidence for del(7)(q22) in uterine leiomyomas.

We analyzed the expression of plasminogen activator inhibitor 1 (PAI-1) in 16 leiomyomas and adjacent myometrium of women who underwent a hysterectomy while in the proliferative (n = 8) and secretory phases (n = 8) of the menstrual cycle. We localized the PAI-1 and its mRNA expression in smooth muscle and vessel endothelial cells of uterine tissues using immunocytochemistry and in situ hybridization. The expression of PAI-1 mRNA was higher in 11 (68.75%) of 16 leiomyomas compared with the adjacent myometrium (leiomyoma/myometrium ratio, 1.4-3.0; mean, 2.045). The leiomyoma:myometrium ratio of PAI-1 mRNA expression did not change during the proliferative (Phase I) and secretory (Phase II) phases of the menstrual cycle. In the remaining five samples, the leiomyoma:myometrium ratio of PAI-1 mRNA expression was close to 1 (0.8-1.2; mean, 0.92). Because the locus of the PAI-1 gene is on chromosome 7q22, we screened for loss of heterozygosity (LOH) in these samples using the PAI-1 marker and D7S471, an anonymous marker 12 cM telomeric to PAI-1. Four of five samples with low leiomyoma:myometrium ratio had LOH for the PAI-1 and/or D7S471 markers. The fifth sample demonstrated a noninformative analysis for these markers but had LOH for the D7S515, D7S666, and D7S518 markers, all centromeric to PAI-1. Because del(7)(q22), associated with a relatively low PAI-1 mRNA expression, can deregulate matrix proteinases and growth factors' activity in leiomyomas, it is conceivable that del(7)(q22) results in heterogeneous leiomyoma biology.

A protein binding site from the murine c-myc promoter contributes to transcriptional block.

Recent studies have revealed that expression of several eukaryotic genes can be regulated at the level of transcription elongation. As a first step to elucidate the mechanism by which transcription elongation is modulated, several groups have identified sequences necessary for transcriptional block within the c-myc gene. These studies indicated that transcriptional block depends not only on sequences surrounding the sites of block, but also on sequences within the promoter: some deletions within the c-myc promoter eliminated transcriptional block and, with chimeric constructs, transcriptional block was observed when some heterologous promoters but not others were fused to the c-myc termination region. Using a chimeric construct containing the H-2Kb major histocompatibility class gene promoter linked to the c-myc first exon, we show that transcriptional block is increased by the addition of a 25 bp DNA sequence from the c-myc promoter. Similar results are obtained whether this sequence is inserted upstream or downstream of the transcription initiation site. We further show that nuclear factors interact with this sequence in vitro. Interestingly, when a mutated version of this sequence was tested, we observed decreased nuclear factor binding in vitro as well as reduced transcriptional block in nuclear run-on transcription assays. These results suggest that interactions of protein factors with specific nucleotide sequences near the transcription initiation site can affect elongation of transcription at sites located further downstream.

Molecular requirements for transcriptional initiation of the murine c-myc gene.

We have identified sequences in the 5' flanking region of the murine c-myc gene's P1 and P2 transcription initiation sites which form specific complexes with nuclear factors of murine and human origin and are also required for normal P1 and P2 usage. Four nuclear factor binding sites were identified within 400 bp 5' of P1 (5'Mf, 5'Mg1, 5'Mg2, and 5'Mg3) and two others within 100 bp 5' of P2 (ME1a1 and ME1a2). The Sp1 transcription factor bound to 5'Mg1 and 5'Mg3 with high affinity and with low affinity to 5'Mg2, ME1a1 and ME1a2 which also bound with high affinity to other factors in crude nuclear extracts. Deletion mutagenesis of sequences 5' of the P1 initiation site revealed that 109 bp encompassing 5'Mg3 and a TATA sequence were sufficient for P1 usage. The ME1a1 binding site 5' of P2 was necessary for maximal P2 activity and the loss of this sequence resulted in enhanced P1 usage. These findings demonstrate that the P1 and P2 initiation sites are independently regulated and that the ME1a1 binding site plays a central role in the normal usage of the c-myc promoters.

Two transient increases in c-myc gene expression during neuroectodermal differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells of the P19 line can be induced to differentiate into neurons, astrocytes and fibroblasts by exposure to retinoic acid (RA), whereas treatment of the EC cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal tissues, including cardiac and skeletal muscle. In RA- but not DMSO-treated cultures, the level of c-myc mRNA underwent two transient increases. The concentration of c-myc mRNA in RA-treated cultures increased 3-fold after 3 h in the presence of the drug, returned to normal by 9 h, increased again 5-fold from 48 to 96 h, and finally decreased below pre-treatment values by 144 h. Increased levels of c-myc protein were observed at the times of elevated c-myc mRNA. Nuclear run-on assays of the c-myc gene and measurements of c-myc mRNA stability indicated that both transcriptional and post-transcriptional mechanisms contribute to the modulated expression of the c-myc gene. The two transient increases in c-myc expression suggest a role for the c-myc protein in the differentiation of cells along neuroectodermal lineages.

Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells.

We have analysed the regulation of c-myc expression in murine fibroblasts and F9 teratocarcinoma cells. The initiation of c-myc transcription is induced to similar levels after serum stimulation of confluent and subconfluent Balb/c A31 fibroblasts while intragenic pausing within the gene's first exon remains unaffected. Sense c-myc transcription continues unabated for at least 18 hours in subconfluent cells, whereas in confluent cells it rapidly falls to pre-induced levels. Cytoplasmic c-myc mRNAs accumulate within 1-2 hours of serum addition to subconfluent cells and reach a higher level than expected from the degree of induction of sense transcription. However, c-myc mRNA levels fall close to pre-induced levels by 18 hours demonstrating that c-myc expression is initially subject to strong positive and then eventually strong negative post-transcriptional control. Anti-sense transcription within the c-myc locus was found to be constitutive under all these physiological states, thereby demonstrating that c-myc transcriptional control is strand specific. Epidermal growth factor stimulates c-myc transcription in a way different from that of serum: (1) initiation of transcription is not significantly enhanced, but intragenic pausing is significantly abrogated; and (2) post-transcriptional mechanisms do not enhance the degree of c-myc mRNA accumulation. In contrast to our results in fibroblastic cells, differentiating F9 teratocarcinoma cells down-regulate c-myc expression entirely at the post-transcriptional level. Our findings indicate that different cell types preferentially employ different modes of myc control depending on their physiological status.

The induction of uterine leiomyomas and mammary tumors in transgenic mice expressing polyomavirus (PyV) large T (LT) antigen is associated with the ability of PyV LT antigen to form specific complexes with retinoblastoma and CUTL1 family members.

The inactivation of certain tumor suppressor genes is thought to play an important role in the genesis of a number of tumor types. For example, inactivation of the Retinoblastoma (Rb) tumor suppressor is frequently observed in a proportion of sporadic human breast cancers. While these studies suggest that inactivation of key tumor suppressor genes may play an important role in the induction of mammary cancers, direct evidence supporting this contention is lacking. Because polyomavirus (PyV) Large T (LT) antigen is known to associate with and inactivate certain members of the Rb family (p105Rb, p107, p130), we have derived transgenic mice which express PyV LT antigen in the mammary epithelium. As expected mammary epithelial-specific expression of PyV LT antigen resulted in the induction of mammary tumors which correlated with their capacity to associate with Rb family members. In addition to mammary carcinomas, female transgenic mice expressing the PyV LT transgene frequently develop uterine leiomyomas. Because loss of heterozygosity involving the human CUTL1 (Cut like 1) gene located at chromosomal position 7q22 has been recently implicated in sporadic human uterine leiomyomas, we tested the hypothesis that PyV LT antigen may also form specific complexes with CUTL1. The results of these analyses revealed that specific complexes of CUTL1 and PyV LT antigen could be detected in both leiomyomas and mammary tumors. Taken together, these observations suggest that PyV LT antigen may be involved in inducing these tumors by sequestering both CUTL1 and Rb growth regulatory proteins.

Refined mapping of the region of loss of heterozygosity on the long arm of chromosome 7 in human breast cancer defines the location of a second tumor suppressor gene at 7q22 in the region of the CUTL1 gene.

In breast cancer, loss of heterozygosity (LOH) has been described on the long arm of chromosome 7, at band q31, suggesting the presence of a tumor suppressor gene in this region. In this study, we have identified a second region of LOH on 7q, at band 7q22. Deletion of genetic material at 7q22 was found in all tumor types and grades and was associated with increased tumor size. The region of LOH at 7q22 in every case included one or more of three polymorphic markers that are located within the CUTL1 gene. LOH of 7q22 has also been documented in the case of human uterine leiomyomas (Zeng et al., 1997; Ishwad et al., 1997). Interestingly, in both leiomyomas and mammary tumors induced in transgenic mice expressing the Polyomavirus (PyV) large T (LT) antigen, immunocomplexes of CUTL1 and PyV LT antigen were detected (Webster et al., 1998). Altogether, genetic data in human breast cancer and biochemical analyses in breast tumors from transgenic mice suggest that CUTL1 is a candidate tumor suppressor gene.

Loss of heterozygosity and reduced expression of the CUTL1 gene in uterine leiomyomas.

Cytogenetic analyses has revealed deletions and/or rearrangments at several chromosomal positions in approximately half of uterine leiomyomas. The most frequent genetic alteration, deletion of 7q22, was found in approximately 35% of studied cases with cytogenetic abnormalities (128/366=35%). The same chromosomal band was also found to be deleted in a fraction of acute myeloid leukemias and myelodysplastic syndromes. The frequent deletion of 7q22 in some tumors suggest that a tumor suppressor gene may be located in this region. The human Cut-like homeobox gene, CUTL1, is one of the genes localized to 7q22 and it was shown previously to encode a transcriptional repressor that down-modulates the expression of c-Myc. Activation of the c-Myc oncogenic potential has been shown in many cancers to result from alterations in one or the other of its several mechanisms of regulation. These observations led us to hypothesize that CUTL1 could act as a tumor suppressor gene. In the present study, we have identified polymorphic markers within and directly adjacent to CUTL1 at 7q22 and demonstrated that these markers are present in a commonly deleted region in seven out of 50 uterine leiomyomas samples examined. Furthermore, Northern blot analysis revealed that CUTL1 mRNA levels were reduced in eight tumors out of 13. These results suggest that CUTL1 may act as a tumor suppressor gene whose inactivation could be of pathological importance in the etiology of uterine leiomyomas.

Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas.

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Cux/CDP homeodomain protein binds to an enhancer in the rat c-mos locus and represses its activity.

The c-mos gene is transcribed in male and female germ cells, in differentiating myoblasts and in 3T3 cells from cell-specific promoters. We characterized the rat testis promoter, which contains a TATA-box and one binding site for a testis-specific transcription factor TTF-D, as well as a region which can act as enhancer, which is located approx. 2 kb upstream of the c-mos AUG start codon. It binds three factors at sites I, II and III as determined in DNAse I footprint assays. We demonstrated that a member of the NF-1/CTF family of transcription factors binds site II. Here we report the cloning of the protein that binds to enhancer site III. This protein is the rat homolog of human hCut/CDP, mouse Cux/CDP and canine Clox. hCut/Cux/CDP/Clox (hereafter called Cux/CDP), a 160 kDa protein containing multiple repeats and a homeodomain, negatively regulates the mammalian c-myc, gp91-phox and N-Cam genes. Using bacterially produced murine GST-Cux fusion proteins and GST-Cux deletion mutants, we find that Cux repeat CR3 and the homeodomain are both required for efficient binding to enhancer site III. Mouse lung and testis nuclear Cux/CDP bind to site III as determined in electrophoretic gel mobility supershift assays using two different anti-hCut specific monoclonal antibodies. Transfections of CAT constructs containing the enhancer fragment linked to a minimal promoter demonstrated that Cux/CDP represses c-mos enhancer activity.

CDP/Cut DNA binding activity is down-modulated in granulocytes, macrophages and erythrocytes but remains elevated in differentiating megakaryocytes.

DNA binding by the CCAAT-displacement protein, the mammalian homologue of the Drosophila melanogaster Cut protein, was previously found to increase sharply in S phase, suggesting a role for CDP/Cut in cell cycle progression. Genetic studies in Drosophila indicated that cut plays an important role in cell-type specification in several tissues. In the present study, we have investigated CDP/Cut expression and activity in a panel of multipotent hematopoietic cell lines that can be induced to differentiate in vitro into distinct cell types. While CDP/Cut DNA binding activity declined in the pathways leading to macrophages, granulocytes and erythrocytes, it remained elevated in megakaryocytes. CDP/Cut was also highly expressed in primary megakaryocytes isolated from mouse, and some DNA binding activity could be detected. Altogether, these results raise the possibility that CDP/Cut may be a determinant of cell type identity downstream of the myelo-erythroid precursor cell. Another possibility, which does not exclude a role in lineage identity, is that CDP/Cut activity in megakaryocytes is linked to endomitosis. Indeed, elevated CDP/Cut activity in differentiating megakaryocytes and during the S phase of the cell cycle suggests that it may be required for DNA replication.

Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development.

CDP/Cux/Cut proteins are an evolutionarily conserved family of proteins containing several DNA binding domains: one Cut homeodomain and one, two or three Cut repeats. In Drosophila melanogaster, genetic studies indicated that Cut functions as a determinant of cell-type specification in several tissues, notably in the peripheral nervous system, the wing margin and the Malpighian tubule. Moreover, Cut was found to be a target and an effector of the Notch signaling pathway. In vertebrates, the same functions appear to be fulfilled by two cut-related genes with distinct patterns of expression. Cloning of the cDNA for the CCAAT-displacement protein (CDP) revealed that it was the human homologue of Drosophila Cut. CDP was later found be the DNA binding protein of the previously characterized histone nuclear factor D (HiNF-D). CDP and its mouse counterpart, Cux, were also reported to interact with regulatory elements from a large number of genes, including matrix attachment regions (MARs). CDP/Cut proteins were found generally to function as transcriptional repressors, although a participation in transcriptional activation is suggested by some data. Repression by CDP/Cut involves competition for binding site occupancy and active repression via the recruitment of a histone deacetylase activity. Various combinations of Cut repeats and the Cut homeodomains can generate distinct DNA binding activities. These activities are elevated in proliferating cells and decrease during terminal differentiation. One activity, involving the Cut homeodomain, is upregulated in S phase. CDP/Cut function is regulated by several post-translational modification events including phosphorylation, dephosphorylation, and acetylation. The CUTL1 gene in human was mapped to 7q22, a chromosomal region that is frequently rearranged in various cancers.

Promoter analysis of the murine T-cell protein tyrosine phosphatase gene.

The T-cell protein tyrosine phosphatase (TC PTP) is expressed ubiquitously at all stages of mammalian development. However, mRNA levels fluctuate in a cell-cycle-dependent manner, reaching peak levels in late G1, and rapidly decreasing in S phase. Furthermore, TC PTP being present in higher amounts in lymphoid tissues, we have recently shown that it is essential for proper maintenance of both the bone marrow micro-environment and B- and T-cell functions. In order to better understand the elements controlling the expression pattern of this gene, we have isolated and characterized approx. 4kb of the murine TC PTP promoter. DNA sequencing of the proximal 5' region revealed the absence of both TATAA and CAAT boxes. Primer extension analysis and S1 nuclease mapping techniques identified multiple transcription initiation sites. Functional promoter activity was determined using transfection experiments of promoter deletion constructs fused to a CAT reporter construct. Our results indicate that the minimal promoter sequence required for functional expression is contained within the first 147bp of the TC PTP promoter. In addition, consistent with the cell-cycle-dependent expression of TC PTP, we localized a domain between 492 and 1976bp from the transcription initiation site through which repression occurs. In conclusion, although initiator-driven transcription allows for ubiquitous expression of TC PTP, we define general transcription motifs present within the promoter that may mediate specific modulations of the TC PTP gene.

Exon/intron structure and alternative transcripts of the CUTL1 gene.

The human CUTL1 gene (Cut-like 1) is a candidate tumor suppressor gene located on chromosome 7 at band 22, a region that is frequently deleted in several human cancers. The gene spans at least 340kb and contains 33 exons. Synthesis of five different transcripts involves two promoter regions, two polyadenylation sites and seven alternative splicing events. The two polyadenylation sites are located at the ends of exons 24 and 33 and are separated by approximately 40kb. Transcription is initiated in two genomic regions, giving rise to alternate first exons which are spliced to a common exon 2. All transcripts contain exons 2 to 14, but differ in their 3' regions. Exon 14 can be spliced alternatively to the beginning or the middle of exon 15, or to exon 25, generating transcripts with exons 15 to 24 or exons 25 to 33. Moreover, exon 16 can be spliced out from the mature transcripts that contain exons 15 to 24. Overall, five distinct transcripts are generated as a result of alternative transcription initiation, splicing and polyadenylation. We discuss potential mechanisms by which alternate polyadenylation site usage may affect alternative splicing events and vice versa.